Tropical , vol. 35, 4, 203-212 (2010) Copyright by the Brazilian Phytopathological Society. Printed in Brazil www.sbfito.com.br

RESEARCH ARTICLE / ARTIGO

In vitro production of conidia of solani

Tatiana T.M.S. Rodrigues, Luiz A. Maffia, Onkar D. Dhingra & Eduardo S.G. Mizubuti

Departamento de Fitopatologia, Universidade Federal de Viçosa, 36570-000, Viçosa, MG, Brazil

Author for correspondence: Eduardo S.G. Mizubuti, e-mail: [email protected]

ABSTRACT Sporulation of Alternaria solani can be scarce and is often reduced when the is cultivated in vitro. A series of experiments were conducted to assess the effects of moisture, mycelial wounding, light quality and photoperiod, and culture media on conidial production. A procedure to induce sporulation based on mycelial wounding and dehydration was adapted and validated. Best results were obtained when fungal colonies were grown in V8 medium at 25oC in the dark with agitation for seven days; the mycelium mass was ground, poured into dextrose agar (pH 6.5) in plates, and incubated at 25 ± 2oC under near ultraviolet light and 12 h-photoperiod. The procedure was validated with 30 isolates of A. solani from different hosts, sampling locations, ages, and storage conditions. Conidial production, germination, and infectivity were quantified. Seventy-five percent of the isolates sporulated and the lowest germination rate was 68%. Inoculations of conidial suspension of all isolates resulted in lesions on and potato plants. The effect of subculturing on A. solani sporulation was also assessed. After six subcultures, every seven days, there was no reduction in sporulation of colonies that were induced with the protocol. Key words: early blight, biphasic technique, subculture, infection frequency.

RESUMO Produção in vitro de conídios de Alternaria solani A esporulação de Alternaria solani in vitro pode ser escassa e tende a reduzir após sucessivas repicagens. Conduziram-se vários experimentos para avaliar o efeito da umidade, injúria da colônia, qualidade de luz e fotoperíodo, bem como do meio de cultura na produção de conídios. Adaptou-se a técnica bifásica para induzir esporulação, por meio de injúria de micélio e desidratação do meio de cultura. Obtiveram-se melhores resultados quando colônias cresceram em meio V8 a 25oC, no escuro, sob agitação durante sete dias; a massa de micélio foi triturada e a suspensão depositada em meio BDA (pH 6,5) em placas de Petri, incubando-se a 25 ± 2oC, sob luz negra com 12 h de fotoperíodo. Validou-se o protocolo para 30 isolados de A. solani distintos quanto ao hospedeiro, local de origem, idade e forma de armazenamento. Quantificaram-se a produção, germinação e infectividade de conídios. Dos 30 isolados, 75% esporularam e a germinação mínima de conídios foi de 68%. Todos os isolados foram patogênicos em seus respectivos hospedeiros. Avaliou-se, também, o efeito de repicagens sucessivas na esporulação. Todos os isolados produziram conídios após seis repicagens sucessivas. Palavras chave: pinta preta, técnica bifásica, subcultura, frequência de infecção.

INTRODUCTION nutrition, light spectrum, and temperature. Natural culture media are suitable for sporulation, storage, and to maintain In vitro experiments are routinely used to study viability of the colony after subcultures (Dhingra & Sinclair, the nature and control of plant diseases caused by fungal 1995). For instance, the V8 juice medium, PDA (potato- pathogens. Sporulation is a key component for several dextrose-agar) and media with parts or extracts of plants are purposes because fungal spores are frequently used as used in protocols to induce sporulation of Alternaria spp. propagules to infect plants. In the case of potato (Solanum (Miller, 1955; Shahin & Shepard, 1979; Dalla Pria et al., tuberosum L.) and tomato (Solanum lycopersicum L.) 1997; Ávila et al., 2000; Vieira, 2004). Commonly, calcium early blight, caused by Alternaria solani Sorauer, conidial carbonate (CaCO3) is also added to increase sporulation suspensions are commonly used as inoculum, but sporulation (Miller, 1955; Shahin & Shepard, 1979), but it is unknown in vitro can be scarce (Rotem, 1994). It is well known that whether its effect is due to the change in pH or due to in vitro sporulation of A. solani requires special conditions calcium supplementation. and that conidial production tends to decrease after periodic Light and temperature affect many aspects of fungal subculturing of the pathogen (Rotem, 1994). development, including the formation of reproductive Considerable attention has been directed towards structures. In vitro sporulation of A. solani is favored by improving conidial production of Alternaria species by incubation under continuous fluorescent light (λ = 380 using unfavorable conditions for vegetative growth (Rotem – 775 ηm) at 25oC or 16 h photoperiod at 20oC (Douglas, & Bashi, 1969; Shahin & Shepard, 1979; Walker, 1980; 1972) and under black light (near ultraviolet – NUV, λ = 320 Ávila et al., 2000; Vieira, 2004). Three major factors that – 400 ηm) and 12 h photoperiod at 25oC (Fourtouni et al., influence sporulation in vitro are commonly manipulated: 1998). However, the spectrum of fluorescent light contains

Tropical Plant Pathology 35 (4) July - August 2010 203 T.T.M.S. Rodrigues et al. inhibitory wavelengths, especially the blue wavelengths mL) and chloramphenicol (75 µg/mL). Isolates preserved (Honda & Nemoto, 1984). Fungal sporulation can be under different methods were used. In this case, colonies either inhibited or stimulated by photoreceptors (Idnurm & were first grown in PDA, in the dark, for 7 days, then two Heitman, 2005). In Neurospora crassa there are at least five plugs of mycelium (5 mm diameter) were transferred to V8 photoreceptor groups, which are related to many aspects of medium in flasks, incubating under agitation (110 rpm) at the fungus life cycle, including sporulation (Purschwitza et 25oC, in the dark. After 7 days, the mycelium was removed al., 2006). and ground using the Polytron® (System PT 1,200 C, The factors described above and stress conditions Handheld Homogenizer), at 4,000 rpm, for 60 s. Ten mL of can be properly combined to induce fungal sporulation. the mycelial suspension was deposited in 15 mL of culture Mycelial injury (wounding) and gradual loss of the culture medium, which varied according to the experiment, in 9 media moisture content lead to stress conditions that cm-diameter Petri plates. Three days after the beginning stimulate sporulation in Alternaria spp., as found with A. of the experiments, 10 mL of DW plus 0.01% Tween 80 macrospora (Walker, 1980), A. cassiae (Walker, 1982; Ávila was used to harvest the conidia with a brush (Foolad et et al., 2000; Pitelli & Amorim, 2003), and A. euphorbiicola al., 2000). The conidial suspension was collected in (Vieira, 2004). In his pioneering work, Walker (1980) beakers; the conidial concentration was determined by described the biphasic technique for massive production of counting the number of spores in a 10 µL-drop under the conidia of A. macrospora. The technique is called biphasic microscope (100X) and multiplying the number counted because initially the mycelium is produced in liquid by 100 to estimate the total number of conidia/mL. The medium, wounded, and then transferred to a solid medium apparatus used to provide light during the incubation to allow sporulation. Little information is available about of the colonies had six light-bulbs spaced 5 cm apart and the mechanism that triggers the production of reproductive located 30 cm above the plates. structures induced by dehydration and wounding. Although many studies were conducted to investigate Effects of culture medium dehydration and mycelium sporulation of A. solani in vitro, in general the results vary wounding on sporulation and the viability and infectivity of conidia taken from Five plates with V8 agar-CaCO3 medium (200 mL ® colonies induced to sporulate were not measured. Another of V8 Vegetable Juice, 3 g of CaCO3, 16 g of agar, 800 problem related to these studies is the low number of isolates mL of DW) per isolate/treatment were used. The V8 agar- assessed. Usually, only a single isolate is used to determine CaCO3 medium was chosen for the first experiment because the favorable conditions to allow in vitro sporulation. This it is a standard medium for A. solani sporulation. Three can be an important issue for highly variable species such treatments were compared: plates sealed with Parafilm®; as A. solani. Therefore, the objective of the present study plates without Parafilm®; and open plates (without the lid). was to adapt the protocol developed by Walker (1980) to Light was provided by three black light bulbs (Sylvania® induce sporulation in A. solani, combining the effects of Luz Negra – 40W - 320-400 ηm) and three fluorescent mycelial wounding, loss of culture media moisture, sources daylight bulbs (Sylvania® Luz do Dia – 40W) with 12 h of light, photoperiod, culture medium, and subculturing. photoperiod. Additionally, the protocol developed was validated with Two isolates (AS073 and AS074) were used in several isolates of the pathogen. the experiment to quantify the effect of culture medium dehydration on sporulation. Each 20 plates/isolate was MATERIALS AND METHODS weighed before and after pouring the culture medium and mycelial suspension. Plates without lids were kept under Isolates, cultural conditions, and conidia quantification black and daylight bulbs, as described above. Every 12 h Thirty different isolates of A. solani were used in the plates were weighed, until 84 h after the beginning of the experiments (Table 1). Isolates preserved on filter paper the experiment. All subsequent experiments used mycelium were used in all tests to adapt the protocol and to assess wounding and plates without lids, to allow for dehydration the effect of subculturing on conidial production. Mycelial and fungal sporulation. disks of monosporic colonies grown in PDA (200 g of potato, 20 g of dextrose, 16 g of agar, 1 L of distilled water- Effects of the source of light, photoperiod, and culture DW) were transferred to Petri plates with PDA covered medium on sporulation with sterilized pieces of filter paper (1 x 1 cm) (Dhingra Three sources of light were compared: daylight & Sinclair, 1995). In all experiments, incubation was at bulbs, black light bulbs, and the combination black / daylight 25oC. After full development of the colonies, the pieces of bulbs. In this last treatment, the bulbs were alternated. Each colonized filter paper were removed and stored in paper source was tested in a continuous light regime or in a 12 o envelopes at -80 C. Two fragments of colonized filter paper h photoperiod. Three plates with V8 agar-CaCO3 medium were transferred to Erlenmeyer flasks containing 150 mL per isolate, per treatment (combination of light source and of V8 medium (200 mL of V8® Vegetable Juice, 800 mL regime) were used. Plates kept under continuous darkness of DW) supplemented with streptomycin sulfate (50 µg/ were used as controls.

204 Tropical Plant Pathology 35 (4) July - August 2010 In vitro production of conidia of Alternaria solani

TABLE 1 - Characteristics of the isolates of Alternaria solani used to develop and/or to validate the protocol to induce sporulation

Isolate Host Location Year Storage(a) AS04-001 Potato Carandaí MG 2004 FP AS073 Tomato Carandaí MG 2004 FP AS074 Potato Domingos Martins ES 2004 FP AS075 Tomato Viçosa MG 2004 FP AS076 Potato Bueno Brandão MG 2004 FP AS077 Potato Ibiá MG 2004 FP AS078 Tomato Venda Nova do Imigrante ES 2004 FP AS079 Tomato Planaltina GO 2006 FP AS082 Tomato Muniz Freire ES 2000 Castellani AS083 Tomato - 1996 Castellani AS088 Potato Ponta Grossa PR 2000 Castellani AS089 Tomato Ponta Grossa PR 2000 Castellani AS090 Tomato Capão Bonito SP 2000 Castellani AS091 Tomato Viçosa MG 2000 Castellani AS094 Tomato Coimbra MG 2000 Castellani AS095 Tomato Coimbra MG 2000 Castellani AS117 Tomato Valão do Barro RJ 2000 Castellani AS121 Tomato Itaperuna RJ 2000 Castellani AS123 Tomato Tocantins MG 2000 Castellani AS124 Tomato Tocantins MG 2000 Castellani AS130 Tomato Cajuri MG 2000 Castellani AS131 Tomato Cajuri MG 2000 Castellani AS132 Tomato Marília SP 2000 Castellani AS223 Tomato Castelo ES 2005 PDA AS250 Potato Conselheiro Lafaiete MG 2005 PDA AS259 Potato Cristalina GO 2005 PDA AS337 Potato Itapetininga SP 2005 PDA AS370 Potato Ipuiúna MG 2006 PDA CE20 Tomato - 1999 SG UFV22FFTomato Viçosa MG 2000 PDA COIMBRA Tomato Coimbra MG 2004 PDA (a) FP = filter paper (-80oC), PDA = tube with PDA (4oC), Castellani method (Dhingra & Sinclair, 1995) and SG = silica gel

Four culture media, amended or not with CaCO3, sporulation was induced according to the protocol were compared: PDA; V8 agar; Vegetable broth agar – VBA developed in the current study. The colonies were grown in (Pereira et al., 2003) and Carrot agar – CA (20 g of carrot, 16 PDA (pH=6.5) in the dark for 7 days. For subculturing two g of agar, 1 L of DW). The pH was adjusted to 7.5 and 6.5 plugs of mycelium taken from the center of each colony in treatments with or without CaCO3 (3g/L), respectively. to establish a new growth were transferred, one plug to Based on the results of the previous experiment, the plates a plate with PDA (pH=6.5) and one plug to a flask with were kept under black light, 12 h photoperiod. Three plates 150 mL of V8 medium. Three plates and three flasks were used per isolate, per treatment. were prepared per isolate at each subculturing. Every 7 days, cultures in plates were subcultured. After colony Effect of periodic subcultures on sporulation development, the cultures in liquid medium were obtained The isolates AS04/001, AS073, AS074, AS075, and induced to sporulate using the best combination from AS077 and AS078 were subcultured and at each transfer, the previous experiments.

Tropical Plant Pathology 35 (4) July - August 2010 205 T.T.M.S. Rodrigues et al.

Validation of the protocol to induce sporulation and Statistical analyses infection A two-way analysis of variance was conducted to Two experiments, the first with 20 isolates and the analyze the data. Whenever the treatment x isolate interaction second with 30 isolates, were undertaken to validate the term was significant, the analyses of the treatment effects protocol. Conidial production was induced in five plates were done by isolate. Each experiment was conducted per isolate. Conidia of three randomly selected plates were twice (hereafter referred to as assays 1 and 2), except those harvested and a 103 conidia/mL-suspension was used in for the quantification of culture medium dehydration, germination and infectivity tests. To evaluate germination, infection frequency, and subculturing. The Levene test was 50 µL of the conidial suspension from each isolate was used to check the homogeneity of variance between assays deposited on one microscope slide. Three slides were conducted twice (Kuehl, 1994). Treatment means were prepared per isolate. In the first experiment, the slides were compared using the Tukey test (α = 0.05). In the experiment transferred to a plastic box (11cm length x 11cm width x with culture media, the pH treatment effect was evaluated 4cm height), kept at 25oC, in the dark, for 12 h, until the by contrasts. In all experiments, the sporulation data were observation under the microscope (100X). In the second transformed to log10 (number of conidia); when there was experiment, the incubation period was 6 h. A conidium was no sporulation in a treatment, the data were transformed to considered as germinated when one germ tube was at least log10 (number of conidia + 1). All analyses were done using as long as the conidium’s width. One hundred randomly the SAS® ver. 8.2. selected conidia were examined per slide. To assess the infectivity of the spores, 45 to 60-day- RESULTS old tomato plants cv. Santa Clara or detached leaflets of potato cv. Monalisa were inoculated. Detached leaflets Effects of culture medium dehydration and mycelium of potato were used because the number of plants was wounding on sporulation not enough to conduct the experiment. Each tomato plant Data from each assay were analyzed separately was divided into three thirds (upper, middle, and lower) because of the lack of homocedasticity. Aerial mycelium based on plant height. In each third, three leaflets were growth with no sporulation was observed in colonies tagged with a paper tag and a cover glass (1 x 1 cm) formed in plates with lids, sealed or not with Parafilm. In was attached on the adaxial surface with double-faced open plates, medium dehydration occurred and vegetative adhesive tape. The conidial suspension was sprayed on mycelia were sparse, and produced asexual structures. The the leaflets using a DeVilbiss 15 sprayer. The negative sporulation varied among isolates, and ranged from 3.72x105 control was sprayed with water and the positive control (SE=0.74x103) to 11.46x105 (SE=3.81x103) conidia/mL and was sprayed with the isolates AS078/AS079 and AS079, from 2.79x105 (SE=0.74x103) to 5.99x105 (SE=2.27x103) in experiments 1 and 2, respectively. For each isolate, conidia/mL, in assays 1 and 2, respectively. The highest rate three plants were inoculated and kept for 24 h in a of culture medium dehydration (45%) occurred in the first moist chamber (100% relative humidity). Nine detached 12 h. After 60 h, no loss of moisture was observed until the leaflets of potato were inoculated as above, and kept last observation at 84 h. in a plastic box. After the inoculation, the cover glass was removed and placed upside down on top of a drop Effects of the source of light, photoperiod, and culture of lactophenol on a microscope slide. The number of medium on sporulation conidia deposited on the cover glass was counted under Data from both experiments were pooled and the microscope (100X). The number of lesions formed in analyzed. Sporulation of all isolates was significantly the leaflets was counted on the third and fourth day after (P<0.0001) affected by light source and photoperiod. inoculation. Based on the results of experiment 1, only Highest number of conidia was observed in colonies kept leaflets of the middle third of tomato or potato plants under black light (P<0.0001) and 12 h photoperiod (Figure were used in experiment 2. Six leaflets in each plant and 1). No isolates sporulated under continuous darkness. four plants/isolate were inoculated. The data from the assays of culture media were analyzed The infection frequency (IF), considered as the separately. The interaction between culture medium ratio between the number of lesions (L) and the number (supplemented or not with CaCO3) x pH was significant in of conidia deposited on a leaflet, was used to assess three of 16 interactions in assay 1, and in 2 of 16 interactions infectivity. Leaflet area was measured on the fifth day in assay 2 (Table 2). Although a high number of conidia after inoculation, using an electronic leaf area meter were produced in all media, either supplemented or not with ® 2 (Li-3.100 – Licor ). The cover glass area (1 cm ) was CaCO3 (Table 3), PDA medium at pH=6.5 was chosen for subtracted from the total leaflet area to result in a corrected the subsequent experiments due to its lower cost. number of spores deposited on a given leaflet CN( ). The IF was determined as: Effect of periodic subcultures on sporulation Every colony sporulated until the 6th subculture. IF(%) = (L/CN)x100. Different colonies of the same isolate did not differ

206 Tropical Plant Pathology 35 (4) July - August 2010 In vitro production of conidia of Alternaria solani

AS04-001 AS073 A a a 4 A A 4 3 b b 3 B 2 B B 2 a 1 a a 1 b

)/mL 0 0 24 hours

(conidia

10 AS074 AS075 a 12 hours A log 4 4 3 3 b 2 a B 2 1 b 1 0 0 DDBB DDBB Light

FIGURE 1 – Sporulation [log10 (conidia)/mL] of Alternaria solani in different sources of light (D = Daylight, DB = Daylight/Black, B = Black) and photoperiod (24 h and 12 h). For each isolate, bars with the same capital letter, for the same source of light, do not differ according to the Tukey test (α = 0.05). Bars with the same small letter, for the same photoperiod, do not differ according to the Tukey test (α = 0.05).

TABLE 2 – F values and, in parentheses, significance level, for differences between media of four isolates of Alternaria

solani in different culture media supplemented (pH=7.5) or not (pH= 6.5) with CaCO3, in two assays (1 and 2)

Culture medium Isolate Assay PDA V8 agar VBA CA All 1 12.58* 1.16 7.11 0.01 6.91 AS04-001 (0.0027) (0.2982) (0.0169) (0.9059) (0.0182)

2 53.60 11.10 4.84 7.86 31.68 (<0.0001) (0.0042) (0.0429) (0.0127) (<0.0001) 1 26.28 16.63 10.64 4.95 16.72 AS073 (<0.0001) (0.0009) (0.0049) (0.0409) (0.0009)

2 17.21 0.38 1.12 14.47 12.85 (0.0009) (0.5443) (0.3070) (0.0017) (0.0027) 1 0.67 0.00 1.60 0.12 0.71 AS074 (0.4246) (0.9628) (0.2244) (0.7297) (0.4122)

2 13.28 8.97 0.03 19.32 29.49 (0.0022) (0.0086) (0.8637) (0.0005) (<0.0001) 1 65.54 0.73 6.77 37.58 29.00 AS075 (<0.0001) (0.4064) (0.0192) (<0.0001) (<0.0001) 2 67.43 10.13 1.38 14.77 49.43 (<0.0001) (0.0058) (0.2570) (0.0014) (<0.0001) * Average from 3 replicates.

regarding sporulation. At least one colony of each isolate Validation of the protocol to induce sporulation and sporulated until the 9th subculture. For AS077 and AS078, at infection least one colony produced conidia until the 18th subculture The following protocol was defined: colonies should and one colony of the isolate AS074 sporulated until the be grown in V8 medium under agitation (110 rpm), at 25oC, 20th subculture (Figure 2). in the dark, for 7 days. The harvested mycelia should be

Tropical Plant Pathology 35 (4) July - August 2010 207 T.T.M.S. Rodrigues et al.

TABLE 3. Sporulation [log10(number of conidia+1)/mL] of four isolates of Alternaria solani in different culture media supplemented (pH=7.5) or not (pH= 6.5) with CaCO3, in two assays (1 and 2)

Culture Media PDA V8 VBA CA pH 1*3 2 1 2 1 2 1 2

*4 AS04/001 6,5 5,04 5,10 4,49 4,87 4,43 4,64 4,35 4,63 A* a A a** A ab A b A b A c A b A c

7,5 4,42 4,71 4,68 4,70 3,96 4,76 4,32 4,49 B a B a A a A a A b A a A ab A b

AS073 6,5 4,98 5,10 4,73 4,89 4,24 4,98 4,27 4,80 A a A a A ab A a A b A a A b A a

7,5 4,26 4,68 4,15 4,82 4,70 5,10 3,96 4,42 B ab B b B b A ab A a A a A b B b

AS074 6,5 4,57 4,48 2,85 4,73 2,77 4,58 2,99 4,05 A a A ab A b A a A b A a A b A b

7,5 4,31 3,98 2,87 4,32 2,36 4,60 3,11 3,44 B a B b A b A ab A b A a A b B c

AS075 6,5 5,24 5,13 4,48 4,58 4,49 3,94 4,77 4,30 A a A a A b A b A b A c A b A b

7,5 3,92 4,40 4,62 4,30 4,91 4,04 3,77 3,96 B b B a A a A b A a A b B b B b * For the same isolate, culture medium and assay, averages followed by the same capital letter do not differ according to the Tukey test (α = 0.05). ** For the same isolate, pH and assay, averages followed by the same small letter do not differ according to the Tukey test (α = 0.05). *3 Assays 1 and 2. *4 Sporulation= log10 (number of conidia + 1)/mL. Average from 3 replicates. ground using Polytron®, at 4000 rpm, for 1 min, and 10 mL Profuse mycelial growth without the production of of the mycelial suspension transferred to plates with PDA conidia is commonly observed in vitro conditions. In (pH=6.5). The plates should be kept without lids, under six general, vegetative growth requires different conditions light-bulbs of black light spaced 5 cm apart and placed 30 from sporulation (Dhingra & Sinclair, 1995). In vivo, cm above the plates; 12 h photoperiod, at 25oC, for 60 h. conidiophores are formed under high humidity and light, In experiment 1, all isolates sporulated, and the whereas conidial formation is favored by alternating minimum germination rate was 96%. All isolates were high-low humidity with darkness (Waggoner & Horsfall, pathogenic to their original host species, but the infection 1969). Simulating the in vivo conditions that favor frequency varied. Greater infection frequency occurred sporulation under controlled conditions has not been with isolates from potato. For most isolates, the highest successful in previous attempts to develop a protocol to and lowest values obtained from the middle and upper third induce sporulation in A. solani. Other factors, besides of the plants, respectively (Table 4). In experiment 2, 23 light conditions and levels of humidity may be required out of 30 isolates sporulated, because seven isolates did to stimulate conidial formation in colonies developed in not grow in liquid medium. In general, the germination vitro. rates in experiment 2 were lower than in experiment 1. The dehydration rate and aeration of the mycelia The methodology used in experiment 2 allowed better affected sporulation of A. solani in vitro. Initially, plates observation of the germinated conidia and more accurate without lid had high humidity, but after 12 h, there counting. In experiment 2 only the middle third of the was a 10% decrease of the initial mycelium mass and plants were inoculated and all isolates were pathogenic to conidiophores were formed under dehydration conditions. their original hosts (Table 5). Mycelial dehydration may trigger the initial stimulus for sporulation. However, the mechanism involved in this DISCUSSION process in A. solani is unknown. It was reported that conidiophores in contact with a film of water may revert The adapted and validated protocol allowed to vegetative hyphae and halt conidiogenesis (Aragaki efficient induction of sporulation of A. solani, in vitro. et al., 1973). In Neurospora crassa, genes associated

208 Tropical Plant Pathology 35 (4) July - August 2010 In vitro production of conidia of Alternaria solani

AS04-001 AS073

6 Colony 1 6 Colony 2 Colony 3

5 5

4 4

3 3

AS074 AS075

6 6

5 5 (conidia)/mL

10 4 4 Log

3 3

AS077 AS078

6 6

5 5

4 4

3 3 1 3 5 7 9 11 13 15 17 19 21 23 1 3 5 7 9 11 13 15 17 19 21 23

Subculture Subculture

FIGURE 2 - Sporulation [log10 (conidia)/mL] of six isolates of Alternaria solani during 24 periodic subcultures. Colonies 1, 2 and 3 are replicates of each isolate. Bars represent standard deviation.

with conidiogenesis were translated only when colony Humidity, CO2, and oxygen levels affect the sporulation of desiccation occurred (Changgong et al., 1997). In the A. solani, but how these factors act on the physiology of the current work, the treatments that did not cause moisture sporulation remains to be elucidated. loss promoted mycelial growth and no sporulation. The protocol used in the present study also resulted Associated with dehydration, greater aeration of in satisfactory sporulation of other species of Alternaria the colonies may also have contributed to pathogen (Walker, 1980, 1982; Ávila et al., 2000, Vieira, 2004). sporulation. Low accumulation of carbon dioxide as well Moreover, it can also be used to induce sporulation in as higher oxygen availability seems to favor sporulation Colletotrichum graminicola, C. malvarum, Drechslera of A. solani (Lukens & Horsfall, 1973). The pathogen is sorghicola, Helminthosporium spp. and Curvularia spp. more sensitive to the increase of carbon dioxide and the (Walker, 1980). This protocol is effective for production of decrease of oxygen during the reproductive rather than massive amounts of inoculum when strict aseptic conditions the vegetative growth phase (Lukens & Horsfall, 1973). are not required (Walker, 1980).

Tropical Plant Pathology 35 (4) July - August 2010 209 T.T.M.S. Rodrigues et al.

TABLE 4 - Sporulation, percent of conidial germination and infection frequency of 20 isolates of Alternaria solani in tomato and potato. Experiment 1, to validate the protocol

Infection frequency(c) Isolate Sporulation(a) Germination(b) Upper third Middle third Lower third AS04/001 8.8 (0.34) 97 (0.67) 0.30(d) (0.18) 13.12 (2.30) 9.26 (3.45) AS073 10.5 (0.91) 98 (0.33) 0.35 (0.11) 1.06 (0.42) 0.17 (0.06) AS074 3.1 (0.16) 96 (0.88) 0.10 (0.05) 3.19 (0.47) 6.40 (2.64) AS075 10.4 (0.53) 98 (0.00) 0.11 (0.07) 0.19 (0.08) 0.07 (0.04) AS076 21.0 (1.20) 99 (0.58) 2.21 (0.54) 2.47 (0.57) 2.27 (0.38) AS077 6.3 (0.17) 86 (2.33) 4.30 (1.24) 7.84 (3.35) 5.77 (2.32) AS078 20.2 (1.35) 99 (0.33) 0.04 (0.02) 0.23 (0.06) 0.13 (0.04) AS079 53.6 (0.30) 96 (0.58) 5.52 (2.33) 2.84 (0.69) 1.75 (0.42) AS088 5.34 (0.34) 99 (0.67) 10.08 (5.54) 2.01 (0.36) 2.92 (0.70) AS089 2.1 (0.15) 97 (1.20) 0.13 (0.08) 0.42 (0.20) 0.05 (0.02) AS124 13.9 (0.55) 99 (0.33) 1.35 (0.22) 2.72 (0.54) 1.23 (0.42) AS131 10.2 (0.39) 90 (0.58) 0.04 (0.02) 0.00 (0.00) 0.03 (0.01) AS132 13.48 (1.00) 96 (1.20) 0.16 (0.06) 0.48 (0.25) 0.56 (0.16) AS091 2.3 (0.06) 96 (0.00) 0.18 (0.03) 0.29 (0.12) 0.25 (0.15) AS095 6.6 (0.23) 99 (0.00) 0.18 (0.05) 0.10 (0.07) 0.12 (0.08) AS121 10.84 (0.49) 99 (0.33) 0.05 (0.02) 0.04 (0.01) 0.12 (0.08) AS123 6.44 (0.19) 98 (0.58) 0.55 (0.27) 0.55 (0.15) 0.14 (0.05) CE 20 8.26 (0.48) 92 (0.33) 0.26 (0.11) 0.10 (0.04) 0.07 (0.02) UFV 22FF 12.48 (1.42) 98 (0.58) 0.02 (0.01) 0.05 (0.02) 0.03 (0.01) COIMBRA 31.58 (1.21) 98 (0.33) 0.05 (0.03) 0.28 (0.08) 0.16 (0.11) Control*- 1 AS078 11.04 (0.34) 98 (0.33) 0.03 (0.02) 0.09 (0.02) 0.07 (0.01) Control- 1 AS079 6.84 (0.15) 98 (0.88) 3.54 (0.79) 3.18 (0.63) 1.44 (0.32) Control- 2 AS078 9.94 (0.34) 98 (0.33) 0.02 (0.01) 0.07 (0.01) 0.07 (0.03) Control- 2 AS079 7.44 (0.19) 98 (0.33) 0.07 (0.02) 0.29 (0.06) 0.20 (0.06) (a) Average of 5 values (conidia x104/mL of distilled water), number in parentheses is the standard error x 104; (b) average of 3 replicates; (c) [(number of lesions/ number of conidia in each leaflet) x 100], the isolates were inoculated on their original host, and (d) average of 9 replicates (%). * Control 1 and 2 = isolates used as positive control.

Both the quality and the amount of light are critical In A. solani, the production of conidiophores has for sporulation of A. solani (Leach, 1962; Douglas, 1972). nutritional requirements distinct from the production of In the present work, greater sporulation was obtained conidia. The fungus requires a source of carbon (sugar) under alternated regime of light (12 h photoperiod). This to produce high quantity of conidiophores, but high alternation simulates conditions required for conidial availability of sugar inhibits the production of conidia formation, as conidiophore formation occurs under (Waggoner & Horsfall, 1969). This fact was observed in irradiation and conidium formation during the dark period the present study when sucrose was added to the V8 liquid (Lukens, 1960; Leach, 1967; Waggoner & Horsfall, 1969; medium: the fungus grew abundantly, but produced only Douglas, 1972; Walker, 1980). Wavelengths near 310 ηm, conidiophores (data not shown). After the pioneering work provided by black light, stimulate sporulation of A. solani of Miller (1955), CaCO3 has been added to various culture and the conidiophores do not revert to vegetative hyphae media, in different protocols, but the results vary according when exposed to the dark, allowing conidial formation to the medium tested. In this work, as in Vieira’s (2004), (Aragaki et al., 1973). Some authors have speculated that sporulation was higher when VBA was supplemented with photoreceptor pigments could be involved in inducing CaCO3, although there were no significant differences from or inhibiting sporulation by certain wavelengths (Leach, the medium without CaCO3. However, sporulation was

1965; Trione & Leach, 1966; Honda & Nemoto, 1984). A lower in PDA supplemented with CaCO3, a fact that has compound called P310 (“Photoreceptor” 310) was obtained been observed previously on A. solani (Moretto & Barreto, from sporulating colonies of A. chrysanthemi grown under 1995). Despite the evidence that the addition of CaCO3 black light, but not from non-sporulating colonies grown increases sporulation, it is not clear whether the effect is due in the dark (Leach, 1965). The authors postulated that this to the change in pH, or the supplementation of calcium. compound could also play a role in sporulation of A. dauci Periodical subculturing, commonly used with (Trione & Leach, 1966). The chemical structure and the fungi, can affect sporulation and pathogenicity of fungal function of P310 have not yet been determined. plant pathogens (Dhingra & Sinclair, 1995). In A. solani,

210 Tropical Plant Pathology 35 (4) July - August 2010 In vitro production of conidia of Alternaria solani

TABLE 5 – Sporulation, germination and infection frequency (number of lesions/ number of conidia in each leaflet) x 100, of 23 isolates of Alternaria solani. Experiment 2, to validate the protocol

Isolate Sporulation(a) Germination(b) Infection frequency(c) AS078 0.72 (0.07) 82 (3.52) 0.40(0.16) AS082 2.10 (0.21) 68 (1.20) 0.17 (0.06) AS083 33.11 (1.25) 79 (1.20) 0.20 (0.05) AS084 0.74 (0.05) 87 (1.20) 0.44 (0.08) AS088 3.20 (0.16) 92 (0.88) 0.45 (0.05) AS089 2.78 (0.13) 80 (1.20) 0.18 (0.02) AS090 0.76 (0.05) 69 (0.58) 0.75 (0.39) AS091 0.70 (0.07) 74 (0.88) 0.17 (0.02) AS094 3.79 (0.15) 79 (3.18) 0.19 (0.04) AS095 12.88 (0.44) 93 (1.16) 1.17 (0.15) AS117 4.38 (0.17) 74 (2.03) 0.06 (0.02) AS121 1.42 (0.09) 82 (0.88) 0.67 (0.08) AS123 1.12 (0.086) 74 (1.86) 0.31 (0.05) AS124 0.84 (0.051) 76 (2.52) 1.02 (0.18) AS130 3.06 (0.09) 90 (0.88) 0.52 (0.12) AS131 1.40 (0.11) 72 (0.88) 0.77 (0.12) AS132 2.40 (0.16) 90 (1.20) 0.59 (0.1) AS223 1.16 (0.12) 87 (1.20) 0.21 (0.05) AS250 7.74 (0.14) 87 (2.52) 0.28 (0.03) AS259 3.82 (0.12) 89 (1.77) 0.25 (0.03) AS337 2.04 (0.09) 89 (4.71) 0.26 (0.03) AS370 2.32 (0.13) 82 (1.77) 0.51 (0.07) Control*- 1 AS079 5.62 (0.15) 89 (2.19) 1.08 (0.14) Control– 2 AS079 6.22 (0.14) 89 (0.58) 0.30 (0.04) Control– 3 AS079 10.56 (0.56) 88 (2.08) 0.57 (0.09) Control– 4 AS079 1.20 (0.11) 83 (1.53) 0.15 (0.05) Control– 5 AS079 3.04 (0.16) 85 (1.86) 0.49 (0.08) (a) Average of 9 values (conidia x104/mL of distilled water), number in parentheses represents the standard error x 104; (b) average of 3 values (%); (c) average of 24 values (%). * Control 1 to 5 = isolate used as positive control.

periodical subculturing affects sporulation (Rotem, 1994). was difficult. In experiment 2, germination rates were In the present study, the way the fungus was subcultured lower than in experiment 1, but the methodology allowed allowed for all isolates to sporulate for at least six better evaluation of germination. Furthermore, conidia subcultures. In another study, after two or three successive were infective to tomato and potato leaflets. Differences subcultures, the isolates of A. solani no longer sporulated in the inoculated material, whether detached leaflets or (Padhi & Rath, 1973). According to the authors, the isolates not, may have influenced the rates of infection frequency. that sporulated longer were mutants that emerged in culture Changes in the physiology of the detached leaflets may after fungal isolation. have resulted in greater susceptibility, therefore leading The production of a good quantity of quality inoculum to high values of infection frequency being observed for is crucial for studying many aspects of fungal species. The potato isolates. Moreover, A. solani has great pathogenic evaluated protocol resulted in proper amounts and quality variability, which may be the main cause of the variations of sporulation of A. solani. The high rate of germination of the infection frequencies (Rotem, 1994). Despite the found in experiment 1 cannot reflect reality. Due to the long variations, all isolates were infective and the protocol incubation time (12 h), the germ tubes with multiple cells developed was appropriate to induce sporulation of A. formed a net of mycelia and identifying individual conidia solani in vitro.

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TPP 9115 - Received 28 August 2009 - Accepted 15 September 2010 Associated Editor: Nilceu R.X. Nazareno

212 Tropical Plant Pathology 35 (4) July - August 2010